The goal of this project is to determine the complete genome sequence of Staphylococcus epidermidis, a coagulase negative staphylococci (CoNS) which is an emerging pathogen of increasing clinical significance. Of the CoNS group, S. epidermidis is the most virulent and the most common isolate from human infections. The infections caused by S. epidermidis include bacteremia, osteomyelitis, pyoarthritis and foreign body infections. Emergence of S. epidermidis as a significant pathogen has been accompanied by a concurrent increase in methicillin resistant isolates (MRSE) and clinical isolates with intermediate resistance to vancomycin. Because S. epidermidis often serves as a reservoir for resistance determinants that are shared with Staphylococcus aureus, the emergence of strains fully resistant to vancomycin undoubtedly represents one of the most frightening challenges in antibiotic resistance. Clearly, the complete genome sequence and a set of recombinant clones would provide a tremendous resource for the study of S. epidermidis. The S. epidermidis strain chosen for this project is RP62A, a well characterized, methicillin resistant, slime producing strain that is pathogenic to humans and virulent in animal infection models. The approach will be a modified whole genome random sequencing strategy successfully used at TIGR to completely sequence eight prokaryotic genomes. The project will consist of four phases: 1) construction of random small and medium insert plasmid libraries and a large insert BAC library from S. epidermidis RP62A, 2) sequencing both ends of approximately 20,250 small insert clones (40,500 sequence fragments), 3) sequencing the ends of a set of minimally overlapping BAC clones and medium size plasmid clones to provide a scaffolding structure that will minimize the effort required for gap closure and provide confirmation of the underlying assembled structure, and 4) assembly and annotation of the genome to identify structural features, assign gene and functional roles to open reading frames based on database similarity searches. This project will complement TIGR's ongoing S. aureus sequencing project and enable us to determine virulence factors unique to each species. This project will enable us to identify the virulence factors, their regulatory networks and pathogenic mechanisms used by S. epidermidis to invade and infect human tissue. The data developed from this study will be deposited into a variety of databases including the TIGR World Wide Web site. In addition, small insert plasmid and BAC clone sets will be made available to the research community.